Olanzapine Protects Against Rotenone-induced Injury On Dopaminergic Neurons Via AMPK/Autophagy Pathway Activation

Objective: To explore the putative protective effects of olanzapine onrotenone-induced injury to dopaminergic neurons, and the possible underlingmechanisms.Methods: Firstly, the cytotoxicity effect of olanzapine on PC12cells wasestimated. by CCK-8assay. The level of p-AMPK and the relevance of autophagicprotein LC3, p62and p70-s6k were determined by Western blot. LC3was also analyzedby immunofluorescence staining. PC12cells were exposed to rotenone （0.5μM） for24hours to establish the in vitro model of PD. Cells were pretreated with20μMolanzapine for24h, exposed to rotenone for another24h, and then subjected toHochest33258staining, CCK-8assay and Western blot. To study the potentialmechanism of neuroprotection, cells were pretreated with compound C （2.5μM）3hours followed by the addition of olanzapine and rotenone. To explore theneuroprotective properties of the olanzapine against rotenone in vivo, fifty-two maleLewis rats were randomly divided into four groups （n=13）. Rotenone was freshlyemulsified in sunflower oil at1.5mg/ml. Rotenone infusion group receivedsubcutaneous injection of rotenone （1.0ml/kg/day） for8weeks, and the samequantity of oil was injected as vehicle to the control group. For the treatment group,0.9mg/kg olanzapine was intragastric administration once daily in addition to the rotenone,and normal saline in the same volume was given as vehicle to the olanzapine group. Thebehavior features were observed. Dopaminergic neurons in the substantia nigra parscompacta （SNpc） were examined with TH immunofluorescence and the expression ofα-synuclein in dopaminergic neurons was double immunofluorescent staining with TH.Results: The results of Western blot revealed that olanzapine activates AMPK andthe peak of p-AMPK/AMPK ratio was at20μM and the best time is48h. Immunofluorescence staining and Western blot analysis showed that the ratio ofLC3-II/I was increased while the protein level of p62and p70-s6k was decreased witholanzapine treatment. The results of the CCK-8assay and Hoechst33258demonstratedthat olanzapine pretreatment markedly increased the percentage of viable cellsfollowing exposure rotenone. Western blot analysis showed olanzapine diminishedrotenone-induced increase of cleaved PARP protein level and reduced the level ofα-synuclein induced by rotenone. Compound C pretreatment blocked the autophagyinduction and attenuated the neuroprotective effects. The results of the in vivo studyindicated that the total score of olanzapine-treatment group was reduced compared withrotenone-group, besides, olanzapine increases TH-positive neurons in the SNpc anddegrades α-synuclein aggregates in the surviving dopaminergic neurons.Conclusions: The results indicated that olanzapine possesses neuroprotectiveeffects both in vivo and in vitro, and this effect may through the activation ofAMPK/autophagy.